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!U! Nedia
ART Equipments
ART Consumables
Frozen Donor Semen Sample
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Sorm Fonclion Tosls
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Reg. Office:
Works:
A-3S, Nirman vihar, Delhi-110092
1-11-1S3, Sindhi Bazar, Jalna - +31203, (N.S.) !ndia
info@cryocellindia.com, cryocellindia@hotmail.com
www.cryocellindia.com
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PRINCIPLE:
Reagents :
Storage and Stability :
Method :
The HOS test is based on the ability oI live spermatozoa to
withstand moderate hypo-osmotic stress. Dead spermatozoa
whose plasma membranes are no longer intact do not show
swelling. Also the dead spermatozoa with intact plasma
membranes have no osmo-regulatory mechanism active and
showuncontrolled swelling resulting in the rupture oI their over
distended plasma membranes.
The spermatozoa that show controlled swelling under test
conditions is considered the good Iraction with low osmotic
Iragility.
1. Cryo-HOS reagent - 1.0 ml in ampoule
150 mOsm solution oI Sodium Citrate and Fructose
o
Store at 2-8 Ctill expiry date. Expiry indicated on label
1. TransIer the number oI ampoules required to run the batch
oI tests to incubator at 37oC. Allowto stabilize temperature Ior
10-15 minutes
CRYO-HOS KI1
1he Hypo-osmotic Swelling 1est
1. 2.
2. Mix 0.1 ml oI liqueIied semen with 1.0 ml reagent in
ampoule and cover with paraIilm
3. Incubate Ior 30 minutes at 37oC. Remix the solution and
transIer one drop oI sperm suspension to clean microscope
slide and mount using 22x22 mm cover-slip.
4. Examine using preIerably phase contrast zoa. (II phase
contrast microscope is not available normal binocular
microscope with reduced lighting can be used equally) Each
spermatozoon is scored as either normal or showing swelling oI
the tail region.
The result oI the test is expressed as the percent swollen cells,
equivalent to the proportion oI osmotically competent
spermatozoa. Osmotically incompetent and dead spermatozoa
burst openand showstraight tails.
Results :
Fructose is a marker Ior seminal vesicle Iunction. Its Iunction in
the seminal plasma is as a substrate Ior the glycolytic
(Anaerobic) metabolism oI the spermatozoa. This is an
important energy source Ior the sperm and exclusion oI the
seminal vesicular component Irom the ejaculate will result in
almost completely immotile sperm.
Fructose levels in semen are androgen dependent. Fructose is
secreted by seminal vesicles. Fructose levels should be
determined in any patient with azoospermia and especially in
those whose ejaculate volume is less than 1 ml, suggesting
seminal obstruction or atresia or ejaculatory tract duct
obstruction. Absence oI Iructose, low semen volume, and
Iailure oI the semen to coagulate indicate either congenital
absence oI the vas deIerence and seminal vesicles or
obstructionoI the ejaculatoryduct.
Disorders oI the seminal vesicles and a subsequent reduction in
the Iructose concentration in semen will also result in a reduced
motility oI sperm. As the seminal vesicles contribute more than
halI oI the total volume oI semen, absence oI seminal vesicular
secretions will reduce semen volume. Thus the measurement oI
Iructose concentration in men with lowvolume ejaculates may
be oI great value diagnostically.
CRYO-FRUC1O-QUAL
Qualitative Fructose Detection Kit
3. 4.
Another situation where Iructose estimates are helpIul is in men
with polyzoospermia and low motility. Occasionally in men
with very high sperm concentrations (more than 350 million
sperm per ml), the sperm are immotile due to relative deIiciency
oI Iructose. II this semen picture is present, the diagnosis can be
conIirmed by Iinding very low concentrations or even absence
oI Iructose in semen.
1. Cryo-Fructo-Qual reagent 20 ml
2. Fructose Standard 5.0 mg/ml 1 ml
1. Test tubes
2. Test Tube Holder
3. Droppers
4. Pipettes
5. Boiling water bath
O
Store at 2-8 Ctill expiry date indicated on label.
1. Take 2 ml Cryo-Fructo-Qual reagent 2 test tubes. Label one
as test(T) andother and standard(S).
2 Add 0.2 ml standard in S tube and 0.2 ml semen sample in T
tube. Mix well.
3 Keep both the tubes in boiling water bath which is already
boiling Ior one minute. Cool by keeping incold water.
4 Examine the color oI both the tubes. Standard will show
CRYO-FRUCTO~QUAL Reagents :
EQUIPMENTREQUIRED
Storage and Stability:
Method :
CRYO-FRUC1O-QUAL
Qualitative Fructose Estimation Kit
5.
pink-red color.
Samples showsing similar or more color than the Standard are
interpreted as positive Ior Iructose. Colorless or very Iaint are
interpretedas negative Ior Iructose.
All human, organic material should be considered potentially
hazardous.
Handle all specimens as iI capable oI transmitting HIV or
hepatitis. Always wear protective clothing when handling
specimens.
The reagent contains Hydrochloric Acid which is highly
corrosive and irritant. Proper saIety gear should be used while
perIormingthe test. The reagent should be properly disposed.
Interpetation :
WARNINGS ANDPRECAUTIONS
6.
Fructose is a marker Ior seminal vesicle Iunction. Its Iunction in
the seminal plasma is as a substrate Ior the glycolytic
(Anaerobic) metabolism oI the spermatozoa. This is an
important energy source Ior the sperm and exclusion oI the
seminal vesicular component Irom the ejaculate will result in
almost completelyimmotile sperm.
Fructose levels in semen are androgen dependent. Fructose is
secreted by seminal vesicles. Fructose levels should be
determined in any patient with azoospermia and especially in
those whose ejaculate volume is less than 1 ml, suggesting
seminal obstruction or atresia or ejaculatory tract duct
obstruction. Absence oI Iructose, lowsemen volume, and Iailure
oI the semen to coagulate indicate either congenital absence oI
the vas deIerence and seminal vesicles or obstruction oI the
ejaculatoryduct.
Disorders oI the seminal vesicles and a subsequent reduction in
the Iructose concentration in semen will also result in a reduced
motility oI sperm. As the seminal vesicles contribute more than
halI oI the total volume oI semen, absence oI seminal vesicular
secretions will reduce semen volume. Thus the measurement oI
Iructose concentration in men with low volume ejaculates may
be oI great value diagnostically.
Another situation where Iructose estimates are helpIul is in men
7. 8.
with polyzoospermia and lowmotility. Occasionally in men with
very high sperm concentrations (more than 350 million sperm
per ml), the sperm are immotile due to relative deIiciency oI
Iructose. II this semen picture is present, the diagnosis can be
conIirmed by Iinding very lowconcentrations or even absence oI
Iructose in semen.
1. Precipitating Reagent, 10 TCA Ior precipitation oI
semen proteins.
2. Color Reagent, Indole-3-acetic acidreagent.
3. Calibrator, Fructose Standard solution 500 mg/dl.
1. Test tubes
2. Test Tube Holder
3. Droppers
4. Pipettes
5. Boiling water bath
o
6. Incubator at 37 C.
7. Colorimeter
O
Store at 2-8 Ctill expiry date indicated on label.
1. Mark one tube Ior calibrator and one tube Ior each sample.
Pipette 0.9 ml precipitating reagent in each tube.
2. Pipette 0.1 ml oI calibrator in calibrator tube and 0.1 ml well
mixed semen sample in each labeled tube. Mix well and allow10
minutes Ior precipitation.
CRYO-FRUCTO~QUANTReagents :
EQUIPMENTREQUIRED
Storage and Stability :
CRYO-FRUCTO~QUANTMethod :
3. CentriIuge all sample tubes at 1500 RPM Ior 5 minutes to
settle the precipitatedproteins andobtainclear supernatant.
4. Label another set oI tubes and transIer 50 micro-liters oI
diluted calibrator and sample supernatant to appropriately
labeled tubes. Add 50micro-liters color reagent inall the tubes. \
5. Add 2 ml concentrated Hydrochloric acid to each tube and
mix well.
0
6. Incubate at 37 C Ior 75 minutes. Keep all the tubes well
coveredwith paraIilm or plastic Ioil.
7. Read absorbance at 520 nanometer or equivalent Iilter on
colorimeter.
8. Calculate usingIollowingIormula
ConcentrationoI test mg/dl
Concentration oI Iructose in semen ranges Irom 63 to 500 mg/dl
(3.5 to 28 mmol/l). It must be remembered that as sample ages;
the Iructose level will Iall due to utilization oI Iructose by
spermatozoa. The more sperm that is present in the ejaculate, the
greater the Iall in Iructose concentration.
All human, organic material should be considered potentially
hazardous.
Handle all specimens as iI capable oI transmitting HIV or
hepatitis. Always wear protective clothing when handling
specimens.
The reagent contains Hydrochloric Acid which is highly
corrosive and irritant. Proper saIety gear should be used while
perIormingthe test. The reagent shouldbe properly disposed.
Assay levels and interpretation:
WARNINGS ANDPRECAUTIONS
Cryocell Sperm Morphology Stain
Staining method for human spermatozoa
For in vitro diagnostic use only
9.
Intended use :
General information :
Material included with the Kit :
Cryocell Sperm Morphology staining kit, is a diagnostic kit Ior
staining human spermatozoa. The purpose oI staining
spermatozoa is to be able to diIIerentiate morphologically
normal Irom abnormal sperm cells.
The deIinition and criteria Ior normality have been largely based
on studies done on sperm recovered Irom the Iemale
reproductive tract (especially in post coital cervical mucus) and
also Irom the surIace oI zona pellucid, which are considered
normal. Now WHO also has accepted strict morphological
criteria Ior classiIication oI sperms ans notmal or abnormal.
Cryocell Sperm Morphology Stain Kit is a rapid aid in
evaluating morphology in a way that it helps distinguish the
diIIerent parts oI the sperm cell (head, acrosome, equatorial
region, midpiece, tail), making it easier to diIIerentiate between a
normal and an abnormal spermatozoon.
Reagent A: red stain - 50ml or 250ml
Reagent B: Blue stain - 50ml or 250ml
Fixative 10 ml in dropper bottle
10.
Material not included with the Kit :
Storage and stability:
Preparation :
Method :
Glassware
Coplin jars
Microscope
Immersionoil
Warm plate at 37C
Tap or distilled water
Cryocell Sperm Morphology stain should be stored in closed
Coplin jars or the original bottles, at 15-25C. The reagents are
stable Ior 36 months aIter date oI manuIacture iI unused.
However, staining removes constituents and introduces
contaminants, and thus stains should be replaced when adequate
stainingis nolonger achieved. Filter stains iI deposit is noted.
Pour the reagents in Coplin jars, make sure the Iluid level is high
enough to cover the area that is to be stained. Fill two Coplin jar,
or any other recipient that can hold the slides, with tap water (Ior
washing the slides between the diIIerent dyes). II the tap water is
alkaline (pH~ 7), thenuse distilledwater Ior washing.
Use washedcleanslides withIrostedendIor makingsmears.
1. Allowa thin Ieathered-edge smear oI Iresh, undiluted semen to
air dry Ior about 5minutes ona warm plate at 37C.
2. Fix the smear by putting Iew drops oI Iixative on horizontal
slides so that the Iixative covers all the areas oI smear. Allowthe
11. 12.
Iixative to evaporate and let the slides dry completely.
3. Stain in stain A using 3-5 dips oI 1 second each. Wash by
dipping 7-10 times in Iresh tap water. BrieIly drain excess water
onto absorbent paper.
4. Stain in stain B using 7-8 dips oI 1 second each. Wash by
dipping 7-10 times in Iresh tap water. BrieIly drain excess water
onto absorbent paper.
5. Allowsmear to air dry.
6. Observe staining under a light microscope (100x) using oil
immersion:
- acrosome bluish pink
- nucleus stained dark blue
- equatorial region pale pink
- midpiece and tail pink
- Count at least 100 and preIerably 200 spermatozoa and classiIy
them as either normal or abnormal, speciIying which deIects are
most common.
- Only include identiIiable sperm cells in the count.
- The criteria Ior classiIying sperm cells as either normal or
abnormal depends on the classiIication method used inthe lab.
Cryocell Sperm Morphology Stain should be stored at room
temperature (15-25C).
Interpretation of the results :
Storage :
Mounting slides :
Warnings and Precautions:
II slides are mounted staining will Iade under mounting medium
(aIter weeks). So do not mount slides iI you want to reIer back
later. Gently blot oII immersion oil, which also Iades the
staining. It is preIerable to make duplicate slides Ior Iuture
reIerence iI necessary, or photographic or videorecords.
- All semen samples should be considered potentially inIectious.
Handle all specimens as iI capable oI transmitting HIV or
Hepatitis.
CRY-JI1ALI1Y E
CRYO-JI1ALI1Y EA
13.
SPERMVITALITYSTAINING KITS
MATERIALINCLUDEDINTHEKIT
STORAGE
The sperm vitality is reIlected in the proportion oI spermatozoa
that are 'alive. It is measured by assessing the ability oI sperm
plasma membrane to exclude extra-cellular substances like dyes.
Sperm vitality should be determined in semen samples with less
than IiIty percent motile spermatozoa. Vitality assessment also
provides check on the accuracy oI motility assessments; as the
percentage oI live spermatozoa should be slightly exceed the
total percentage oI motile spermatozoa.
Eosin-Nigrosin staining is used Ior assessing vitality. The
technique is based on the principle oI dead cells will take up the
eosin, and as a result stain pink. The Nigrosin provides a dark
background, which makes it easier to assess the slides. The
assessment can be carried out at any time and slides also can be
preserved Ior Iuture assessment and record.
Plain eosin staining also can be used to assess vitality in wet
smears. This provides quick assessment at the same time oI count
and motility assessment.
Cryo-Vitality EN 1 ml
Cryo-Vitality E 1 ml
o
Store reagents at 2-8 C
14.
EQUIPMENTREQUIRED
METHOD
Cryo-VitalityE
Light microscope (1000XmagniIication)
Microscope slides pre-cleaned
Cover slips
Pipettes
Test tubes
Cryo-VitalityEN
1. Mix equal volumes oI well-liqueIied semen and Cryo-
Vitality EN (small drops) on a pre-cleaned microscopic slide.
Allowabout 30 seconds Ior staining.
2 Make smear on the slide using other slide as spreader. Smear
should be thin enough Ior allowing proper visualization. Thick
smears are useless Ior evaluation.
3 Allow air-drying and preIerably mounting with DPX or
equivalent mountant as soon as possible. These slides can be
stored at ambient temperature indeIinitely.
4 At least 100 or preIerably 200 spermatozoa are examined at
a magniIication oI 1000x or 1250x under oil immersion using
high quality non-phase-contrast objective and correctly adjusted
bright-Iield optics.
1 Mix equal volumes oI well-liqueIied semen and Cryo-
Vitality E (small drops) on a pre-cleaned microscopic slide.
Cover with a 22x22 mm cover-slip II there is excess mixture oI
semen and stain seen blot lightly with tissue paper. Allow 5
minutes Ior settling and staining oI the sperms.
CRY-ACD KI1
1he Auclear Chromatic Decondensation 1est
(ACD 1est)
16.
Principle :
Material included with the Kit :
Material not included with the Kit :
Storage and stability :
The chromatin is spermatozoa is in a highly condensed state
prior toIertilization.
The appropriate nuclear chromatin decondensation and
subsequent male pronucleus Iormation is essential Ior
Iertilization andnormal zygote development.
Highly condensed nuclear chromatin is spermatozoa is because
oI the Iormation oI S-S cross links between its histone units. The
cleavage oI S-S link can be induced in vitro by Sodium Dodecyl
Sulphate and EDTA. Such induced decondensation is predictor
oI goodIertilizing oI spermatozoa.
1. NCDIncubation reagent 10x1ml vials/amps
2. NCDDry reagent disks 10nos
3. Fixative StainReagent 2ml Dropper Bottle
Incubator Ior 50C
Glassware
Microscope slides and cover slips
Microscope
Store at 2-8Ctill expiry date. Expiry indicatedonlabel
15.
2 Examine under high power using high quality non-phase-
contrast objective and correctly adjusted bright-Iieldoptics.
Spermatozoa that are white (unstained) are counted as live and
those showing any degree oI pink or redare dead.
Spermatozoa stained with this kit can not be used Ior any Iurther
procedures. To diIIerentiate live spermatozoa Irom dead
spermatozoa Ior use in ICSI (low viability samples), the HOS
(hypo-osmotic swelling) test should be used.
All human, organic material should be considered potentially
hazardous.
Handle all specimens as iI capable oI transmitting HIV or
hepatitis. Always wear protective clothing when handling
specimens.
INTERPRETATION
LIMITATIONS OFTHEMETHOD
WARNINGS ANDPRECAUTIONS









17. 18.
Method :
1. Pipette 0.5 ml oI NCDIncubation reagent in small plastic vial
with cap. Add one disk oI NCDDry reagent to the pipette reagent
and shake to let the disk elute.
2. Incubate the vial at 50C Io 5-10 minutes to achieve
equilibration at this temperature.
3. Add 100 microlitre oI well mixed semen sample to the
incubation mixture and incubate Iurther Ior 5 exactly.
4. At the end oI incubation time add on drop oI Iixative and one
drop oI stain reagent to the incubation mixture.
5. Mix well and take a small drop on slide and place a 22x22 mm
cover-slip.
6. Examine under lowpower and then high power oI microscope.
The cells showing swelling oI nucleus with uniIorm chromatin
are considered positive and the cells showing intact nucleus are
considered negative NCD.
Result :
The result oI the test is expressed as the percent swollen cells oI
NCDreactedcells to toal sperms.
More than 70 Sperms in normal sample showNCDpositivity.
Less than 70 NCD positivity is associated with reduced
IertilityoI the sperm.
Warningand Precautions :
All semen samples should be considered potentially inIectious.
Handle all specimens as iI capable oI transmitting HIV or
Hepatitis.